CN101921336A - Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof - Google Patents

Abstract

The invention aims to prepare a specific monoclonal antibody aiming at alpha1 acid glycoprotein (AGP, Serum alpha1-acid glycoprotein) and establish a corresponding experimental method. Particularly, the monoclonal antibody can selectively identify glycosylation modified natural AGP, but has low affinity with prokaryotically expressed non-glycosylation modified AGP. The subtype of the monoclonal antibody is IgG1, and the affinity constant is 9*108. The invention provides a hybridoma cell line BPI-AGP for secreting the monoclonal antibody, wherein the collection number of the hybridoma cell line is CGMCC 2868, and the hybridoma cell line can stably secrete high-valence monoclonal antibody against AGP natural protein. The BPI-AGP can be used for inspecting the AGP in the blood. Meanwhile, the invention also provides a method for preparing monoclonal antibody hybridoma aiming at the glycosylation modified protein.

Description

Monoclonal antibody of a kind of anti-people α 1 acid glycoprotein and preparation method thereof

Technical field

The invention belongs to immunochemistry, protein chemistry and cytobiology field.Particularly, the present invention relates to a kind of hybridoma cell line that can produce special monoclonal antibody at people α 1 acid glycoprotein (AGP, Serum α 1-acidglycoprotein) and preparation method thereof.The monoclonal antibody of this hybridoma preparation can be applied to the diagnosis or the association area of disease.

Technical background

Proteinic posttranslational modification state and protein function are closely related, and wherein glycoprotein has been brought into play multiple important biological function, as the glycoprotein on the cytolemma participate in that fetal development, cell are moved, processes such as immune response and cell fission.In addition, most of plasma proteins all has in various degree glycosylation modified.It should be noted that known protein as various diseases certification mark thing all is glycoprotein now, as PSA, AFP, CA125, CA153 and HER-2 etc.For many years, obtained certain progress about research to the detection of the biomarker in the blood plasma.Wherein most important method is produced exactly at these proteic antibody, and the method by ELISA realizes these proteic detections and quantitatively.The key of this method promptly is to obtain good, the highly sensitive antibody of specificity.At present, the method of traditional production antibody is quite ripe and in most of the cases very effective, but production for these glycoprotein antibody in the blood plasma, often there is certain deficiency in traditional method, this is to be difficult to acquisition owing to have the albumen of Natively glycosylated modification, and the antigenicity of glycoprotein is often not strong.The albumen of prokaryotic expression, glycosylation modified owing to not having, the antibody of taking this to produce often can not be discerned the glycoprotein in the human blood well.The production of glycoprotein antibody can adopt traditional antibody production method to carry out Antibody Preparation by enrichment and the intravital natural sugar albumen of purifying biological as antigen usually.Content is lower in vivo but this method is for those, and the glycoprotein that perhaps is difficult to purifying is also improper.And the more important thing is in a series of manipulation in vitro processes, whether the various character of glycoprotein are caused in various degree influence, also be unknown.Therefore in order to overcome above difficulty, some scientists have have researched and developed the preparation method for antibody that makes new advances to obtain special antibody at glycoprotein.McKenzie adopts mouse peritoneal to be injected at the method for the tumour cell of stable expression of exogenous glycoprotein Neu on its cytolemma as immunizing antigen, and the stimulation body produces and filters out at this proteic monoclonal antibody.In the method, the Neu gene is imported in the NIH3T3 cell, make its stably express on cytolemma.After cell was gone into mouse by abdominal injection, this proteic extracellular part can produce antibody as the antigenic stimulation body.This method has overcome the difficulty that glycoprotein on the film is difficult to enriching and purifying, and experimental result also shows, the antibody that the antibody that this method is produced is produced as antigen than the peptide section that relies on the synthetic Neu albumen has better specificity, can distinguish the albumen high with its similarity.Atabai also arrives in the rabbit body by the tumor cell injection of similar method with the proteic people of stably express MFGE8, obtains monoclonal antibody specific by screening.Suzuki etc. are connected to carrier proteins KLH upward as antigen with the sugar chain of heparin, adopt traditional method to be expelled in the mouse body, but filter out the monoclonal antibody of this sugar chain of specific recognition.But above-mentioned research can not provide a kind of applicability method widely, and the experimental implementation more complicated.

The inventor is an example with people AGP, but attempts to set up the scheme of the antibody of a cover production specific recognition glycosylated protein, and the monoclonal antibody of Sheng Chaning can overcome glycoprotein antigen lowly and the defective of glycoprotein antibodies specific difference basically thus.The glycoprotein gene of external source is imported among the mouse melanin tumor cell B16 by retroviral vector, obtain the stably express strain through screening.This expresses strain can arrive the extracellular with the exogenous sugar protein excretion constantly.Stable cell line and adjuvant mixed after be subcutaneously injected in the C57BL/6J mouse body, along with the continuous propagation and the growth of cell, exogenous sugar albumen is produced antibody as the antigenic stimulation body by continuous release in the mouse body.After mouse boosting cell that serum is positive and myeloma cell are merged,, but can obtain the proteic monoclonal antibody of specific recognition exogenous sugar by repeatedly screening.This method is compared with traditional production glycoprotein monoclonal antibody method has remarkable advantages: in this process, cell expressing excretory albumen has correct glycosylation modified, do not need they are carried out enrichment, and these antigens are being secreted in the mouse body of persistence, constantly stimulate the immunity system of body, so both solve the difficult problem of glycoprotein enrichment, solved the low problem of glycoprotein antigen again.We adopt this method successfully to produce the monoclonal antibody of AGP, and these antibody can the glycosylation modified AGP of specific identification, and nonglycosylated AGP is had recognition capability hardly.

Summary of the invention

One) the present invention relates to a kind of mouse resource monoclonal antibody BPI-AGP that can specific recognition plasma glycoprotein AGP, and the application of this monoclonal antibody in disease detection.The subclass of this monoclonal antibody BPI-AGP is the IgG1 type, and can the glycosylation modified AGP of specific identification, and nonglycosylated AGP is had recognition capability hardly.

Two) the present invention relates to a kind of hybridoma cell line that can secrete specific recognition AGP monoclonal antibody.The anti-AGP monoclonal antibody BPI-AGP that this hybridoma cell line produces is used for the detection to the expression level of blood glycoprotein.As the abundance that is used for detecting blood samples of patients AGP under the morbid state changes.

Three) the present invention relates to prepare the method for this hybridoma cell line, comprising: 1) will contain the retroviral vector transfection virus packing cell of the foreign gene AGP that has the RFP label, collect viral postoperative infection B16 cell, and stablize the screening of strain by drug treating, stablize the secretion of strain by the RFP antibody test to these glycoprotein; 2) with the stable strain of gained with after Freund's complete adjuvant mixes, through being subcutaneously injected into the flank place of C57BL/6J mouse spleen side; 3) using Freund's complete adjuvant to carry out abdominal part hypodermic reacts with enhancing immunity; 4) AGP of end user's plasma proteins and prokaryotic expression screens simultaneously to monoclonal antibody, and is special at glycosylation modified proteic monoclonal antibody to obtain; 5) to the detection and the assessment of these monoclonal antibodies.

Four) the present invention has selected secretory protein as antigen, and this antigen can be secreted into the extracellular, directly into blood, stimulates body to produce humoral immune reaction.Different with traditional antibody production method, this antigen be constantly continue along with the growth of cell to be secreted into extracellular.Therefore, the stimulation that causes also is successional.Aspect cell and host's selection, melanoma cell B16 promptly is host C57BL/J6 mouse source, therefore, the mouse of having injected stable strain cell only has immune response to ectogenic secretion glycoprotein, and can immune response not arranged to other any albumen in B16 cell self source.This strategy has guaranteed the unicity and the superiority of the antibody produced.And the B16 cell be a kind of easy transfection, cell that foreign gene can stability and high efficiency be expressed.

Five) the present invention has considered the factor of three aspects simultaneously to the introducing of label protein red fluorescent protein RFP: 1) because the RFP gene is the C end that merges at external source glycoprotein, therefore, it can not exert an influence to proteic secretion, and in the process of whole transfection, infection and screening, red fluorescence can show that transfection, efficiency of infection and foreign protein are in intracellular expression situation; 2) when the stable strain that screens was detected, RFP antibody can be used as an effective instrument, and the secretion of external source glycoprotein is measured; 3) as a kind of carrier proteins, RFP can increase the immune response of human body to glycoprotein.This principle is similar to and adopts KLH to react as the carrier proteins enhancing immunity when using polypeptide as immunogen.This is because the sugar chain on the glycoprotein has reduced the antigenicity of glycoprotein as a kind of haptens, therefore uses the bigger RFP of molecular weight (28Kd), rather than usually the HIS-Tag etc. of usefulness as fusion rotein.

Six) the present invention tends to select to adopt retrovirus as carrier foreign gene to be imported in the B16 cell.This is because concerning the bigger glycoprotein of some mrna lengths, with the RFP gene fusion after the excessive method that is unfavorable for adopting traditional direct transfection of mrna length stablize the screening of strain.And retrovirus can the bigger gene segment of packing ratio, and its efficiency of infection is often very high, can shorten screening greatly and stablize the required time of strain.But the present invention equally also relates to the application of other transfection methods in this antibody producing process.

Seven) the present invention adopts the subcutaneous injection immunity at spleen side flank place.This mainly is to consider the following aspects: 1) at first the immunoreactive vitals of the interior generation of body promptly are spleens; 2) in numerous immunization wayses, antigenic direct spleen immunization ways has been proved to be a kind of effective choice more.But this injecting method is remarkable for the influence of body, causes the death of animal probably.In the present invention, because B16 is a kind of grade malignancy height, the easy tumour cell that shifts, the very easy death that causes mouse.If in B16 cell direct injection mouse spleen, then in very short time, just may cause the death of mouse, and also not a large amount of generation of potent antibodies this moment.

Eight) the present invention initiates adjuvant and cytomixis injection.Crucial effect is played in the use of adjuvant in the antigen presentation process, can improve production of antibodies greatly.In the present invention, after adjuvant and the cytomixis injection, though can kill the part cell, after in a single day the cell of survival secreted glycoprotein, the adjuvant around it will in time be presented these antigens, increased production of antibodies.Our experiment also shows that the hybrid injection of adjuvant can obviously improve the output of antibody.And behind injection cell, injection also is in order to strengthen the immune response of human body under the independent adjuvant abdomen.

Nine) technology of manufacture order clonal antibody involved in the present invention is applicable to producing at the antibody of striding the outer albumen (as the cell matrix proteinoid) of film and film.This is that the stimulation body that these parts that are arranged in extracellular albumen or albumen can be used as the antigen persistence produces antibody owing to after changing these genes over to the B16 cell.The antibody that this method produces is often only discerned the extracellular region territory of transmembrane protein, has stronger specificity.And these stride the outer albumen of film and film and often also have certain glycosylation modifiedly, therefore, but use this method can produce these glycosylation modified antibody of specific recognition.Use this method also can produce special antibody, comprise phosphorylation, nitration etc. at other modified forms of protein.As long as the albumen of modification can take place under native state, can both produce by this method can the proteic antibody of this modified forms of specific recognition.

Description of drawings

Fig. 1: the immunoblotting detected result of strain secretory protein stablize in expression, and the result shows that the glycoprotein that has the RFP fusion tag successfully is secreted into the extracellular, and it is tangible glycosylation modified to show that also AGP has taken place from the molecular weight variation of target protein.

Fig. 2: expression B16-RFP-AGP stablizes the fluoroscope detected result of strain, and particularly, the fluorescence micrograph of B16-RFP-AGP shows that red fluorescence only is distributed in the kytoplasm zone, and this has also confirmed the secretion character of AGP glycoprotein.

Fig. 3: injecting immune B16-RFP-AGP stablizes the immunoblotting detected result of the mouse resisting anteserum of strain cell, concrete as seen, immunity comprised in the mouse resisting anteserum of B16-RFP-AGP stable cell lines protein glycosylation be modified with the polyclonal antibody that specific identification is inclined to.

Fig. 4: expression BPI-AGP monoclonal antibody and Sigma-AGP monoclonal antibody are to the experimental result of the antigenic immunoblotting difference identification of different modifying state AGP, particularly, the BPI-AGP monoclonal antibody is to the glycosylated protein specific recognition, and the sugar based modified AGP of prokaryotic expression is not discerned, proteic identification also significantly weakens to AGP after the desugar.And the Sigma-AGP monoclonal antibody does not have skewed popularity to the proteic identification of these two kinds of different modifying forms.

Fig. 5: expression BPI-AGP monoclonal antibody and Sigma-AGP monoclonal antibody are to the experimental result of the difference of the antigenic ELISA detected result of different modifying state AGP, particularly, the BPI-AGP monoclonal antibody only has special combination to glycosylation AGP, and the non-glycosylated AGP of prokaryotic expression is not almost had combination.And the Sigma-AGP monoclonal antibody does not all have evident difference to both combination.

Fig. 6: the immunohistochemical experiment result of expression BPI-AGP monoclonal antibody and Sigma-AGP monoclonal antibody, this result shows: the BPI-AGP monoclonal antibody is higher than the identification of Sigma-AGP monoclonal antibody to the glycosylation AGP in the human liver organization to the specificity of the identification of the glycosylation AGP in the human liver organization.

Embodiment

The gene clone of embodiment 1 glycoprotein

The inventor designs the PCR primer according to the gene order (SEQ ID NO.1) of the people AGP that has announced:

AGP 5 ' forward primer

5’-GCCAAGCTTATGGCGCTGTCCTGGGTTCT-3’(SEQ?ID?ON.2)

AGP 3 ' reverse primer

5’-GGCGGATCCTAGGATTCCCCCTCCTC-3’(SEQ?ID?ON.3)

With human liver cDNA library is template, and pcr amplification goes out total length AGP gene, (PCR parameter: 95 ℃ 5 minutes; 72 ℃ of 54 ℃ of 30s of 94 ℃ of 30s 1 minute, totally 40 circulations; Extended after 72 ℃ 10 minutes) be connected with the pLNCX2-RFP plasmid behind Hind III and BamH I double digestion that (the RFP gene comes from the pDsRed2 plasmid, after the RFP gene being downcut with Hind III and Not I restriction endonuclease from this plasmid, be connected on the pLNCX2 of same double digestion plasmid), chemical conversion competent cell E.coli DH5 α, choose mono-clonal extraction plasmid and check order, the checking sequence is errorless.Structure, screening and the checking of the proteic cell strain of embodiment 2 stably express justacrine exogenous sugars

The pLNCX2-RFP-AGP plasmid that extraction builds from E.coli DH5 α and the plasmid pVSV-G of encoding hiv reverse transcriptase envelope protein, (11668-019, amount Invitrogen) is the cotransfection that 1: 5 ratio is carried out two kinds of plasmids according to DNA and transfection reagent Lipofectamine2000.Under the help of chloroquine, two kinds of plasmids are changed among the retrovirus incasing cell GP2-293.Change liquid after 8 hours to remove the toxic action of chloroquine pair cell.After removing 48 hours behind the chloroquine, collect the substratum supernatant of GP2-293, the retroviral particle that contains foreign gene that is released out in the substratum of this moment is maximum.The retroviral particle that precipitates by long high speed centrifugation (4 ℃, 50,000g, 90 minutes) enrichment.After these viruses are carried out renaturation, join in the substratum of target cell B16, (H9268, Sigma) under the help of reagent, these viruses can infect the B16 cell, simultaneously foreign gene are imported in the target cell at Polybrene.Metainfective cell under the screening effect of G418, the time through about 2 weeks, can obtain mono-clonal and stablize the strain cell.Adopt the method for immunoblotting that the stable strain that screening obtains is verified.Method is as follows: after will stablizing the perfect medium sucking-off of strain cell, wash cell 3 times with physiological saline, change serum free medium then into and continue to cultivate 12 hours.Collecting these serum free mediums, is that (UFC803008 Millipore) carries out proteinic concentrate in the substratum for the Amicon Filter of 30kD by molecular weight cut-off.Substratum after concentrating 40 times can be directly used in immunoblotting and detect.Fig. 1 has shown that stable strain is secreted into the immunoblotting detected result of extracellular glycoprotein and the fluorescence micrograph of the stable strain (B16-RFP-AGP) that Fig. 2 shows.The glycoprotein that as can be seen from Figure 1 has the RFP fusion tag is secreted into the extracellular smoothly, and tangible glycosylation modified from showing that also AGP has taken place on the molecular weight of target protein changes.As can be seen from Figure 2, the fluorescence micrograph of B16-RFP-AGP shows that red fluorescence only is distributed in the kytoplasm zone, and this has also confirmed the secretion character of AGP glycoprotein.

The preparation of embodiment 3AGP recombinant antigen

Gene order design PCR primer according to the people AGP that has announced:

AGP 5 ' forward primer

5’-GCCGGATCCATGGCGCTGTCCTGGGTTCT-3’(SEQ?ID?ON.4)

AGP 3 ' reverse primer

5’-GGCAAGCTTCTAGGATTCCCCCTCCTC-3’(SEQ?ID?ON.5)

With human liver cDNA library is template, pcr amplification goes out total length AGP gene, after reclaiming, glue carries out BamH I and Hind III double digestion, after the recovery, be connected with the pET28a carrier of same double digestion, chemical conversion competent cell E.coli DH5 α chooses the positive colony bacterial strain through double digestion checking and plasmid order-checking.Plasmid chemical conversion competent cell BL21 (DE3) with the positive colony chosen.Inoculation can be expressed the BL21 bacterial strain of AGP, when the OD of bacterium liquid value reaches 0.6 left and right sides, adds 1mmol/L IPTG and induces 4 hours.Induce later bacterial strain centrifugal after ultrasonic, go up cleer and peaceful precipitation and do not carry out the SDS-PAGE electrophoresis, the result shows that expressed products is an inclusion body protein.A large amount of culturing bacterium, IPTG induces after 4 hours and receives bacterium, ultrasonication, centrifugal, abandon supernatant, in precipitation, add the inclusion body protein that the extracting solution dissolving that contains 8M urea is expressed.Behind the recentrifuge, supernatant promptly can be used for purifying.Use the Ni-NAT affinity column that the AGP albumen of expressing is carried out purifying, pillar through balance, go up sample, drip washing after, use 10,20, the 50mM imidazoles carries out stepwise elution, removes foreign protein wherein, adopts 100mM imidazoles wash-out target protein at last.Albumen behind the purifying detects the proteic purification effect of AGP through the SDS-PAGE electrophoresis.The preparation of the recombinant protein that other are used has also been experienced identical expression and purge process as RFP.

The preparation of embodiment 4 anti-people AGP monoclonal antibody hybridomas (BPI-AGP, on January 14 2009 preservation day, preserving number is CGMCC 2868, contains the ground common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms)

Stably excreting is expressed the B16 cell of RFP-AGP after trysinization and stopping, centrifugal collecting cell.These cells are after PBS washing three times, and are resuspended with the substratum of serum-free, carry out cell counting.(100 μ L: ratio 100 μ L) is blown and beaten several times mixing repeatedly with ten thousand cells of 5-10 and Freund's complete adjuvant according to equal-volume.Mixture behind the mixing arrives the subcutaneous of mouse spleen side flank place through injector to inject.After, carried out immune strengthening every 7 days at mouse web portion subcutaneous injection Freund's complete adjuvant.

After one month, get blood, the antiserum(antisera) in the blood is carried out immunoblotting detect by eye socket.Select the AGP of procaryotic cell expression, glycosylation modified AGP (G9885 in the human blood, Sigma), the RFP of glycosylated RFP-AGP albumen of B16 stably express strain excretory and procaryotic cell expression and the RFP-AGP mouse resisting anteserum after as Detection of antigen injection tumour cell tires.The about 200ng of sample carries out 12% polyacrylamide gel electrophoresis on every kind of albumen, and electrophoresis carries out electrotransfer after finishing immediately, and process is as follows: pvdf membrane is placed 5mL methyl alcohol, handled 5 minutes; Add 4 times of volumes (20mL) water, accelerate slowly to shake,, remove liquid methyl alcohol stepwise dilution to 20%; Again with changeing film damping fluid (20% methyl alcohol, 25mM Tris-base, 192mM Glycine) balance 15 minutes.Gel is soaked in the commentaries on classics film damping fluid that contains 0.01%SDS 10 minutes.According to blackboard-sponge-filter paper-gel-pvdf membrane--the order of filter paper-sponge-blank, carry out the sandwich folder, put into the Trans-Blot Cell commentaries on classics film instrument (Bio-Rad that 0.01%SDS changes the film damping fluid is housed, Hercules, U.S.A.), 350mA changeed film 2 hours.The further treating processes of pvdf membrane is as follows: film soaks with PBST, add PBST and prepare the sealing of 5% skim-milk, shook on the room temperature shaking table 2 hours, 4 ℃ are spent the night, PBST adds the mouse resisting anteserum that dilutes at 1: 2000 after washing film, hatched under the room temperature 2 hours, and washed two anti-(using the HRP mark) of the sheep anti-mouse igg that adds 1: 4000 behind the film, hatched 1 hour, PBST washes film once more, use ECL enhancement type immunoblotting detection kit (RPN2132, GE Healthcare) luminous, the exposure of X sheet.Fig. 3 is the immunoblotting detected result of having injected the mice serum behind the stable strain cell.Immunoblot experiment result (Fig. 3) shows: but at injecting immune produced the antibody of the fusion rotein of identification tag albumen RFP and RFP-AGP prokaryotic expression in the mice serum of stable strain cell, simultaneously, but this polyclonal antibody is the glycosylation modified albumen of specific recognition also, comprises naturally occurring in B16-RFP-AGP be secreted into the outer generation of born of the same parents glycosylation modified AGP and the human blood glycosylation modified AGP having taken place.And this antibody is to the not identification of sugar based modified AGP of prokaryotic expression.This shows that this polyclonal antibody has had glycosylation modified albumen is had specific identification tendency.

Detect tiring of mouse polyvalent antibody with the method for Western blot, promptly can be used for further fusion experiment as the AGP that under the higher situation of dilution in 1: 2000 or Dilution ratio, can detect in the 0.5 μ L human plasma.Get the spleen that immunizing potency meets the requirements of the C57BL/6J mouse, be placed on the cell sieve and pulverize, merge through PEG1500 in 5: 1 ratios and SP2/0 cell.Cell after both having merged is cultivated with the methylcellulose gum semisolid medium, screens through HAT, chooses 500 monoclonal cells and continues to cultivate in 96 orifice plates.Use the RFP of prokaryotic expression purifying and human serum protein (the Albumin/IgG Removal Kit that has removed albumin and immunoglobulin (Ig), Cat.122642, CALBIOCHEM), the substratum supernatant of these monoclonal cells is carried out ELISA filter out and discern different antigenic antibody respectively respectively as the antigen wrapper sheet.The positive colony cell that obtains is injection mouse production ascites earlier, carries out frozen then.

Embodiment 5 Purification of Monoclonal Antibodies

From-80 ℃ of refrigerators or liquid nitrogen container, take out frozen pipe rapidly; Put into water-bath rapidly and stir fast, make frozen storing liquid in 2 minutes, all be melted into liquid.With the frozen pipe of 75% alcohol wipe.In the 15mL centrifuge tube, add the 3mL blood serum medium, frozen storing liquid is sucked centrifuge tube, 1500 rev/mins, centrifugal 5 minutes.Abandon supernatant, hanged cell, be incubated in 6 orifice plates or the bottle with perfect medium.Substratum is 3mL in 6 orifice plates, changes liquid, and supplies 3mL again in second day; The culturing bottle substratum is 5mL.The logarithmic phase cell washs with serum free medium and hangs; Count about 5 * 10 ⁵, 1mL.The cell intraperitoneal injection of mice that suspends.Generally just can begin to collect ascites after 7-10 days, can repeat to get, only finally can collect 1-10mL/ every 2,3 days.Each ascites of taking out 4000 is changeed, and 10 minutes centrifugal, and the centre is an ascites.Careful sucking-off ascites is collected in the centrifuge tube 4 ℃ of preservations.

Under 4 ℃ of conditions, 10000 left the heart 10 minutes with ascites, removed lipid material.Supernatant is drawn in centrifugal back, and dilutes with 1: 3 (ascites, coupled liquor ratio) with coupling buffer, crosses 0.22 μ m film, with HiTrap Protein A FF (17-5079-01, GE Healthcare) monoclonal antibody purification.With going up sample behind the coupling buffer balance pillar of 5-10 column volume.Wash post with the coupling buffer of 5 times of column volumes again.In collection tube, add 60-200 μ L neutralizer, be beneficial to the biological activity of keeping antibody, avoid the antibody inactivation.With 5 times of column volume elution buffer wash-out antibody, and be collected in the collection tube of step 6.Can obtain the higher monoclonal antibody of purifying.These antibody are named as BPI-AGP antibody.

The mensuration of embodiment 6 monoclonal antibody affinity costants

Bag by immunity with the glycosylation AGP that is purified in the human serum (G9885, Sigma) and the AGP of prokaryotic expression purifying, wrapping by concentration is 2 μ g/mL, 100 μ L/ holes, 4 ℃ of bags are spent the night, 1 * PBST washes 3 times.Every hole adds 37 ℃ of sealings of 200 μ L confining liquids 2 hours, and 1 * PBST washes 3 times.Anti-AGP monoclonal antibody (BPI-AGP) and commodity monoclonal antibody (A5566, Sigma), since 1: 200 2 times of gradient dilution, the contrast that blanks of last 1 hole was hatched 1 hour for 37 ℃, and 1 * PBST washes 3 times.Sheep anti mouse two dilutions in anti-1: 20000 of HRP mark, every hole 100 μ L were hatched 1 hour for 37 ℃, and 1 * PBST washes 3 times.Colour developing liquid 100 μ L/ holes colour developing 10 minutes, 50 μ L/ hole stop buffer termination reactions.Measure light absorption value with microplate reader.

(A is B _Max/ 2 o'clock antibody dilution multiple)

150000 is single IgG antibody molecular value, and antibody original concentration unit is mg/mL.The result shows, the commodity monoclonal antibody (A5566, Sigma) to glycosylation AGP (G9885, Sigma) and the affinity costant of the non-glycosylated AGP of prokaryotic expression be respectively: 3 * 10 ⁹With 2.3 * 10 ⁹, and the BPI-AGP monoclonal antibody is respectively the affinity costant of the non-glycosylated AGP of glycosylation AGP and prokaryotic expression: 9 * 10 ⁸With 1 * 10 ⁴

Embodiment 7 monoclonal antibody subclass are measured

By sheep anti-mouse igg to 0.5 μ g/mL, every hole adds 100 μ L, 4 ℃, spends the night with 100mM PBS (pH7.4) dilution bag.The sky liquid that inclines is washed 3 times with the PBS that contains 0.05% tween, and every hole adds 200 μ L confining liquids, hatches 1 hour for 37 ℃.The sky liquid that inclines cleans 3 times with PBS-T.Every hole adds 0.1mL hybridoma supernatant, hatches 1 hour for 37 ℃.The sky liquid that inclines cleans 3 times with PBS-T.With the sheep anti mouse of confining liquid 1: 1000 dilution HRP mark (κ, λ) sheep anti mouse of antibody or 1: 2000 dilution HRP mark (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) the every hole of antibody 0.1mL adds respectively in the suitable hole, 37 ℃ with hatching 1 hour.The sky liquid that inclines cleans 3 times with PBS-T.Every hole adds 50 μ L substrate solutions, surveys the OD value under the 405nm wavelength in 10-20 minute.

Experimental result shows that monoclonal antibody of the present invention is an IgG1 type mouse resource monoclonal antibody.Checking-the immunoblotting of embodiment 8 monoclonal antibody purposes of the present invention

Select the AGP of procaryotic cell expression, glycosylation modified AGP (G9885 in the human blood, Sigma) and the AGP after the desugar can detect the antibody of monoclonal antibody of the present invention and commodity respectively as antigen (A5566 is Sigma) in the specificity to different antigen recognition.

The desugar process is as follows: with the human blood AGP protein dissolution of 50 μ g in the ammonium bicarbonate buffers of the 20mM pH 8.0 of 45 μ L.The denaturing soln that adds 5 μ L 0.2%SDS and 100mM beta-mercaptoethanol then.100 ℃ are heated 10 minutes with the glycoprotein sex change.Behind the cool to room temperature, add the TRITON X-100 of 5 μ L 15%, mixing.After adding 5 μ LPNGase F mixings at last, 37 ℃ of incubations 3 hours.5 minutes termination reactions of 100 ℃ of heating.

The immunoblot experiment process is as follows: the about 200ng of sample on every kind of albumen, carry out 12% polyacrylamide gel electrophoresis, and electrophoresis carries out electrotransfer after finishing immediately, and transfer process is with embodiment 4.Protein transduction is moved on on the pvdf membrane 350mA constant current transferase 12 hour in the ice bath.Crossover process is as follows: film soaks with PBST, add PBST and prepare the sealing of 5% skim-milk, shook on the room temperature shaking table 2 hours, 4 ℃ are spent the night, PBST adds 1 after washing film: 2000BPI-AGP monoclonal antibody or 1: 10,000 commodity monoclonal antibody (A5566, Sigma), hatched under the room temperature 2 hours, and washed two anti-(using the HRP mark) of the sheep anti-mouse igg that adds 1: 4000 behind the film, hatched 1 hour, PBST washes film once more, use ECL enhancement type immunoblotting detection kit (RPN2132, GE Healthcare) luminous, the exposure of X sheet.That Fig. 4 shows is the result of the immune marking of two kinds of monoclonal antibodies, as can be seen from Figure 4, the BPI-AGP monoclonal antibody has special identification to glycosylated protein, glycosylation modified AGP taken place as naturally occurring in, B16-RFP-AGP be secreted into the outer generation of born of the same parents glycosylation modified AGP and the human blood.And this antibody is not discerned the sugar based modified AGP of prokaryotic expression, and proteic identification also significantly weakens to AGP after the desugar.And the monoclonal antibody of commodity does not have skewed popularity to the proteic identification of these two kinds of different modifying forms, as the proteic identification of AGP after naturally occurring sugar based modified AGP that glycosylation modified AGP and prokaryotic expression taken place and the desugar in the human blood is not all had evident difference.

The checking of embodiment 9 monoclonal antibody purposes of the present invention-ELISA experiment

(G9885 Sigma) is used for detecting the difference of BPI-AGP antibody and commodity AGP antibody respectively as antigen for AGP that is purified into from the prokaryotic expression product and the glycosylation AGP that is purified into from human blood that buys from Sigma.The antigen of every hole 200ng, 4 ℃ of bag quilts that spend the night.PBST solution is washed 2 times, 37 ℃ of sealings of 2%BSA solution 2 hours.PBST solution is washed 2 times, the different dilution of PBS gradient one anti-(1: 200,1: 400,1: 800 ... 1: 256000) 37 ℃ hatched 1 hour.PBST solution is washed 3 times, and the anti-mouse two of the HRP mark of dilution in 1: 20000 resists 37 ℃ and hatched 1 hour.PBST solution is washed 3 times, adds chromogenic substrate TMB and develops the color 2M H ₂SO ₄Termination reaction.The absorbance value that 450nm reads such as Fig. 5.Can find out obviously that from Fig. 5 the BPI-AGP monoclonal antibody only has special identification to glycosylation AGP, it is 1: 50 to tiring of glycosylation AGP (200ng), 000, the non-glycosylated AGP of prokaryotic expression almost there is not identification.And commodity AGP antibody does not have evident difference to both identification, tires all at 1: 100 about 000.Checking-the immunohistochemical experiment of embodiment 10 monoclonal antibody purposes of the present invention

The wax disk(-sc) of tissue slice is after the dimethylbenzene dewaxing, and again through 100%, 95%, 85%, 75% ethanol carries out aquation.0.3%H ₂O ₂Room temperature 10 minutes is removed in-house horseradish peroxidase activity, and PBS washed 3 times * 5 minutes.5% skim-milk, 37 ℃ were sealed 2 hours.Drip one anti-(BPI-AGP antibody and commodity monoclonal antibody) (1: 500), wet box spends the night for 4 ℃, and PBS washed 3 times * 5 minutes, and drip sheep anti mouse two and resist, incubated at room 60 minutes, PBS washed 3 times * 5 minutes, DAB-H ₂O ₂Colour developing, mirror is controlled 3-5 minute down, the PBS rinsing, color development stopping, Hematorylin was redyed 45 seconds, 1% hydrochloride alcohol color separation, warm tap water flushing is anti-blue, 75%, 85%, 95% and 100% ethanol dehydration each 15 minutes, dimethylbenzene is transparent, the neutral gum mounting.As shown in Figure 6, Fig. 6 shows that BPI-AGP antibody and commodity AGP antibody are little to the Recognition Different of the glycosylation AGP in the human liver organization, but the specificity of BPI-AGP antibody is better.

Sequence table

＜110〉BeiJing HuaDa protein Research Center Co., Ltd

＜120〉a kind of anti-people α 1 acid glycoprotein monoclonal antibody and preparation method thereof

<130>

<160>5

<170>PatentIn?version?3.5

<210>1

<211>606

<212>DNA

<213>human

<400>1

atggcgctgt?cctgggttct?tacagtcctg?agcctcctac?ctctgctgga?agcccagatc 60

ccattgtgtg?ccaacctagt?accggtgccc?atcaccaacg?ccaccctgga?ccggatcact 120

ggcaagtggt?tttatatcgc?atcggccttt?cgaaacgagg?agtacaataa?gtcggttcag 180

gagatccaag?caaccttctt?ttacttcacc?cccaacaaga?cagaggacac?gatctttctc 240

agagagtacc?agacccgaca?ggaccagtgc?atctataaca?ccacctacct?gaatgtccag 300

cgggaaaatg?ggaccatctc?cagatacgtg?ggaggccaag?agcatttcgc?tcacttgctg 360

atcctcaggg?acaccaagac?ctacatgctt?gcttttgacg?tgaacgatga?gaagaactgg 420

gggctgtctg?tctatgctga?caagccagag?acgaccaagg?agcaactggg?agagttctac 480

gaagctctcg?actgcttgcg?cattcccaag?tcagatgtcg?tgtacaccga?ttggaaaaag 540

gataagtgtg?agccactgga?gaagcagcac?gagaaggaga?ggaaacagga?ggagggggaa 600

tcctag 606

<210>2

<211>29

<212>DNA

<213>primer

<400>2

gccaagctta?tggcgctgtc?ctgggttct 29

<210>3

<211>26

<212>DNA

<213>primer

<400>3

ggcggatcct?aggattcccc?ctcctc 26

<210>4

<211>29

<212>DNA

<213>primer

<400>4

gccggatcca?tggcgctgtc?ctgggt?tct 29

<210>5

<211>27

<212>DNA

<213>primer

<400>5

ggcaagcttc?taggattccc?cctcctc 27

Claims (15)

1. monoclonal antibody is characterized in that described monoclonal antibody is is that the mouse hybridoma cell that January 14, preserving number in 2009 are CGMCC 2868 is that BPI-AGP produces by preservation day.

2. the immunohistochemical methods reagent that contains the described monoclonal antibody of claim 1, it is used for detecting the differential expression of people's liver cancer tissue and healthy tissues AGP.

3. the western blot reagent that contains the described monoclonal antibody of claim 1, it is used for detecting the expression of human blood AGP.

4. contain the enzyme linked immunological absorption reagent of the described monoclonal antibody of claim 2, it is used to detect the expression of the total AGP of human blood.

5. the purposes of the described monoclonal antibody of claim 1, it is used for preparing the purposes of the immunohistochemical methods reagent of the differential expression that detects people's liver cancer tissue and healthy tissues AGP.

6. the purposes of the described monoclonal antibody of claim 1, it is used for preparing the purposes of the western blot reagent of the expression that detects human blood AGP.

7. the purposes of the described mono-clonal of claim 1 dragon antibody, it is used for preparing the purposes of the enzyme linked immunological absorption reagent of the expression that detects human blood AGP.

8. the method that detects of the described monoclonal antibody of claim 1, it comprises that the monoclonal antibody of using claim 1 contacts with biological sample.

9. a cell strain B16 who has a foreign gene agp by injecting immune prepares monoclonal antibody method, it is characterized in that described method for preparing monoclonal antibody is with after Freund's complete adjuvant mixes, through being subcutaneously injected into the flank place of C57BL/6J mouse spleen side with the stable cell line B16 of gained.

10. the described MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 9 is characterized in that employed injecting immune cell strain is the tumour cell of close relative's animal-origin.

11. the described MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 9 is characterized in that described foreign gene agp has the RFP label.

12. the described MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 9, it comprises that those are positioned on the cytoplasmic membrane, though can not be secreted into outside the born of the same parents, has partial sequence to reach the outer albumen of born of the same parents.

13. in the described MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 9, it comprises the production of the monoclonal antibody specific of other posttranslational modification formal proteins except that glycosylated protein.

14. in the described MONOCLONAL ANTIBODIES SPECIFIC FOR method of claim 9, it comprises the method for using this thinking to produce specific antibody by other animals except that the C57BL/6J mouse.

15. a mouse hybridoma cell is BPI-AGP, preserving number is CGMCC 2868, it is characterized in that described mouse hybridoma cell strain stably secretes the described monoclonal antibody of claim 1.

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